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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Dev Dyn. Author manuscript; available in PMC 2010 June 1.
Published in final edited form as:
PMCID: PMC2865133
NIHMSID: NIHMS180707

Toward defining the phosphoproteome of Xenopus laevis embryos

Abstract

Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context.

Keywords: phosphorylation, Xenopus, embryo, proteomics, mass spectrometry

INTRODUCTION

Many proteins are regulated by the reversible phosphorylation of specific serine, threonine, or tyrosine residues, including proteins that control the diverse and complex cellular processes that govern vertebrate development. For example, during Xenopus embryogenesis, the β-catenin protein preferentially accumulates in the nuclei of cells destined to form the dorsal and anterior structures of the embryo. Regulated phosphorylation of specific serine residues at the N-terminus of the β-catenin protein in response to Wnt signaling are required for this spatially controlled accumulation of β-catenin (Amit et al., 2002; Liu et al., 2002). Phosphorylation of CPEB (Cytoplasmic Polyadenylation Element Binding), a protein that binds to the 3’ Untranslated Regions (UTRs) of maternal mRNAs, leads to the translational activation of these stored mRNAs in response to the appropriate developmental cues (Hake and Richter, 1994; Mendez et al., 2000; Sarkissian et al., 2004). A myriad of other examples exist that establish phosphorylation as a universal mechanism for controlling the functions or levels of proteins and the activity of key cellular pathways.

Xenopus embryos have served as a powerful model for examining the role of protein phosphorylation in the pathways and processes governing early vertebrate development. However, the studies of many important phosphorylation events described in the literature were guided initially by discoveries made in other systems. That is, although phosphorylation plays clear and important roles during the development of Xenopus embryos, only a small number of the phosphorylation events examined in this model organism have been identified directly from studies of this organism. Given the probability that many more phosphorylation events critical to vertebrate development remain undiscovered, a large and functionally unbiased data set identifying both phosphoproteins and the precise sites of their phosphorylation would provide a powerful foundation for future functional studies. Here we begin to define the breadth and potential temporal and spatial dynamics of protein phosphorylation within the context of developing Xenopus embryos using recent advances in mass spectrometry and protein preparation that enable the accurate identification of phosphopeptides.

The methodological advances that facilitate the identification of specific phosphopeptides from complex mixtures include the use of metal-ion chromatography to enrich phosphorylated peptides and improvements in peptide fragmentation. In particular, the development of an electron-based dissociation method, electron capture dissociation (ECD) (Zubarev et al., 1998), and more recently the related electron transfer dissociation (ETD) (Coon et al., 2004; Syka et al., 2004; Coon et al., 2005a; Coon et al., 2005b) has allowed efficient cleavage of peptide backbone bonds without regard to phosphorylation and that leave phosphorylated residues intact (Good and Coon, 2006; Chi et al., 2007; Good et al., 2007; Khidekel et al., 2007; Lecchi et al., 2007; Molina et al., 2007; Swaney et al., 2007; Phanstiel et al., 2008). Thus, the new methods allow both the phosphopeptide and the precise site(s) of phosphorylation to be identified.

We used ETD to identify 1,441 different phosphorylation sites present on 654 different proteins from Xenopus oocytes, eggs, and embryos. Additional analysis of gastrula stage embryos identified phosphorylation sites on an additional 541 proteins. The Xenopus phosphoproteins were involved in a diverse array of cellular functions. In particular, many of the identified phosphoproteins are known regulators of embryonic development including translational regulatory proteins, signaling proteins and transcription factors. Our results provide a data-rich foundation for exploiting the experimental advantages of Xenopus to examine regulatory phosphorylation events that direct vertebrate embryonic development and cellular function(s).

RESULTS AND DISCUSSION

Xenopus phosphopeptide analysis with tandem mass spectrometry

To identify Xenopus phosphoproteins and phosphorylation sites, proteins from oocytes, eggs, and embryos were analyzed (Fig. 1A). Proteins were extracted from oocytes, eggs, stage 7 and stage 10.5 embryos (Fig. 1B) and treated with trypsin to generate peptides. The peptides were modified to create their corresponding methyl ester derivatives (Ficarro et al., 2002). This peptide modification serves to eliminate non-specific binding during subsequent steps. Phosphopeptides were isolated by immobilized metal affinity chromatography (IMAC) (Ndassa et al., 2006; Ficarro et al., 2002) and separated by reversed-phase chromatography before elution into the mass spectrometer by electrospray ionization (Martin et al., 2000). The mass spectrometer was set to continuously cycle through the acquisition of a full MS spectrum followed by subsequent ETD-MS/MS scans of the ten most abundant phosphopeptide cations observed. The resulting spectra were analyzed using the Open Mass Spectrometry Search Algorithm (OMSSA) (Geer et al., 2004) to identify the relevant phosphopeptides. Identified peptides with an e-value greater than zero were validated with a target-decoy searching method (Käll et al., 2008) and identified phosphopeptides with less than 5% false discovery rate were included in this current analysis. We report all hits at this false discovery rate to include potentially relevant regulatory proteins, and we feel it is warranted given the potential for functional analysis and validation in Xenopus. In addition, we have partitioned the data such that phosphopeptides with less than 0.5% false discovery rate have been indicated (SI Table 1).

Figure 1
Phosphoproteomic analysis of Xenopus oocytes, eggs and embryos

In our initial analyses the predominant phosphoproteins identified were vitellogenins; the abundant phosphoproteins stored in the cytoplasm of oocytes, eggs, and embryos for nutritive purposes during development. The presence of the vitellogenins prevented the efficient identification of other phosphoproteins. Therefore, we added another step to our protocol. Specifically, prior to trypsin treatment, the vitellogenins were removed using Freon (Fig. 1C) (Evans and Kay, 1991).

Identification of phosphoproteins from Xenopus oocytes, eggs and embryos

Protein samples from Xenopus oocytes, eggs, stage 7 and stage 10.5 embryos were analyzed to identify 1,441 sites of phosphorylation from 1,133 phosphopeptides derived from 654 proteins (Fig. 1B, Table 1, SI Table 1). As predicted from proteomic studies in other systems (Olsen et al., 2006) the most prominent phosphorylation events occurred on serine (76% frequency) followed by threonine (21% frequency). Tyrosine phosphorylation events were rare, constituting only 2% of the sites identified (Table 1). These percentages did not change significantly over the course of development. Most of the phosphopeptides possessed a single phosphorylated residue (Table 1). However, 22% of the peptides contained at least two and as many as six phosphorylated residues (Tables 1 and 3).3). Such multi-site phosphorylations often occur in regulatory domains of proteins that must integrate multiple signaling inputs (Gingras et al., 1999; Liu et al., 2002; Zeng et al., 2005). For example, we observed that a peptide derived from the APC protein was simultaneously phosphorylated on six closely spaced residues (Table 3). APC is a component of the Wnt signaling pathway and activation of Wnt signaling leads to the phosphorylation of APC on multiple residues (Brocardo and Henderson, 2008).

Table 1
Identification of Xenopus phosphoproteins from oocytes, eggs and embryos Phosphorylation events sorted by stage
Table 3
Multiphosphorylated peptides.

Some of the earliest mechanisms that regulate embryonic patterning (Weaver and Kimelman, 2004; Thisse and Thisse, 2005) occur during the gastrula stage of Xenopus development. Our interest in the regulation of these events (Lane and Sheets, 2000; Mitchell and Sheets, 2001; Lane et al., 2004; Mitchell et al., 2007) motivated us to perform additional analysis of proteins from gastrula stage embryos manipulated to perturb embryonic patterning. In particular, we analyzed phosphoproteins present in organizer and non-organizer cells manually dissected from gastrula embryos (Table 2). Also, we analyzed the phosphoproteins from embryos treated with LiCl that enhances Wnt signaling and anterior development, and proteins from embryos injected with a β-catenin morpholino that disrupts Wnt signaling (Table 2). From these analyses we identified an additional 541 Xenopus phophoproteins (Table 2, SI Table 1).

Table 2
Identification of Xenopus phosphoproteins from oocytes, eggs and embryos

Notably, many of the Xenopus phosphorylation events we identified corresponded to those observed in protein orthologs from mice and humans (Fig. 2, Tables 4 and 5).5). For many of these proteins multiple lines of analysis, including cell-labeling experiments, the use of phospho-specific antibodies and mutation of the specific amino acids were used to demonstrate that these specific residues were phosphorylated primarily in the context of cultured cells (see Table 4, column 4). For example, serine 369 (S-369) of the Xenopus RSK-2 kinase was identified as a site of phosphorylation in our analysis (Fig. 2). The same residue in the mouse RSK-2 protein is phosphorylated and this modification triggers RSK-2 activation in response to specific signaling events (Hauge et al., 2006; Hauge et al., 2007).

Figure 2
Evolutionary conservation of Xenopus phosphorylation sites
Table 4
Evolutionary conserved Xenopus phosphorylation sites and events*
Table 5
Validation of Xenopus phosphorylation sites from the analysis of human and mouse proteins

In addition several residues of the Xenopus 4E-BP2 protein were identified as sites of phosphorylation in our analysis (Tables 3, ,44 and SI Table 1). 4E-BPs inhibit translation by binding eIF-4E translational initiation factors (Gingras et al., 1999). Studies in cultured cells indicate that the binding of 4E-BPs to initiation factors is controlled by phosphorylation. Our analysis demonstrated that the Xenopus 4E-BP2 was phosphorylated at several different sites, analogous to known phosphorylation sites in human and mouse 4E-BP2 proteins (Table 4). As an independent method to detect 4E-BP2 phosphorylation events, we analyzed 4E-BP2 from oocytes, eggs, stage 7 and stage 10.5 embryos by protein immunoblotting. Analysis with an antibody that recognizes 4E-BP2 when it is phosphorylated at either Thr33 or Thr42 indicated that 4E-BP2 is phosphorylated to varying extents at all stages examined (Fig. 3, lanes 1-4). Analyzing the same filter with an antibody that detects 4E-BP2 regardless of its modification state revealed the presence of non-phosphorylated protein (Fig. 3, lanes 5-8). These results together with the data summarized in Table 4 increase confidence in the data obtained with mass spectroscopy.

Figure 3
Phosphorylation of Xenopus 4E-BP2

Identified phosphoproteins represented a wide range of expression levels

A major challenge of proteomic analysis using mass spectrometry arises because of the extremely large dynamic range of protein levels expressed in specific cells. Many regulatory proteins such as signaling proteins and transcription factors are expressed at low levels. The detection of such important regulatory molecules can be occluded by the presence of much more abundant proteins in a sample. To determine whether our results were heavily biased towards the detection of abundant proteins we used the Xenopus tropicalis EST (expressed sequence tags) resources (http://www.sanger.ac.uk/Projects/X_tropicalis/) (Gilchrist et al., 2004). Xenopus tropicalis ESTs from different stages of development have been organized into clusters in which each cluster represents the ESTs from a single mRNA. The number of ESTs in a cluster provides an estimate of relative mRNA abundance and therefore, at present and by inference, the best approximate estimate of protein levels. For example, the CIRP2 RNA binding protein is encoded by an abundant mRNA that is highly expressed and the CIRP2 EST cluster contains hundreds of ESTs. In contrast, the EST clusters representing mRNAs expressed at low levels, such as the denticleless mRNA, contain fewer than ten ESTs. Xenopus tropicalis mRNAs and EST clusters were identified for 73 of the phosphoproteins we identified from gastrula embryos (SI Tables 2 and 3.). Notably, the mRNAs encoding Xenopus phosphoproteins were predominantly from EST clusters representing the non-abundant and moderately abundant mRNAs (Fig. 4). These data provided evidence that the Xenopus phosphoproteins we identified were encoded by mRNAs with varying levels of expression and were not enriched for the most abundant mRNA class.

Figure 4
Identified phosphoproteins represented a wide range of expression levels

The Xenopus phosphoproteins represented a diverse array of functional categories

Functional annotation revealed that the identified Xenopus phosphoproteins were involved in a diverse array of cellular processes (Fig. 5). In addition, many of the identified phosphoproteins encoded proteins of unknown function (42%). Of note, was the substantial number of proteins that were categorized as “hypothetical.” These data make it clear that many of these proteins are indeed bona fide proteins expressed in living embryos, underscoring a value in this analysis beyond presenting the functionally unbiased information about the Xenopus phosphoproteome.

Figure 5
Functional annotation of Xenopus phosphoproteins

Many of the Xenopus phosphoproteins function to regulate processes that play a central role in vertebrate embryogenesis (Table 6, SI Table 3). For example, in oocytes, eggs and early stage embryos normal development depends heavily on post-transcriptional processes such as mRNA translation and mRNA localization. Consistent with the notion that such processes must be robustly regulated in developing embryos, phosphorylation sites were identified on several different Xenopus proteins involved in translation and RNA metabolism (Table 6, SI Table 3). For example, the translational regulatory protein embryonic poly (A) binding protein (ePABP) was phosphorylated at multiple sites (Table 6, SI Table 3). ePABP binds to mRNA poly (A) tails to stimulate translation and protect mRNAs from degradation (Wilkie et al., 2005). Interestingly, serine 255 of ePAB was phosphorylated in eggs; this serine residue resides within one of the ePABP RRM domains important for poly (A) binding, raising the possibility that phosphorylation may regulate ePABP's ability to bind poly (A). The identification of this and many other previously unexamined phosphorylation sites sets the stage for future functional studies in a wide variety of research areas relevant to vertebrate embryogenesis and cell function.

Table 6
Developmentally relevant Xenopus phosphoproteins Phosphorylation of developmentally relevant proteins*

Conclusion

The data presented here provides an information-rich foundation for investigating the role of specific phosphorylation events by exploiting the many established experimental advantages of Xenopus oocytes, eggs, embryos and in vitro extracts. Despite the large number of undocumented phosphorylation sites uncovered in this study, the data presented here remains some distance from saturation. Further developments in mass spectrometry technology that couple high mass measurement accuracies with ETD promise an even deeper exploration of the Xenopus phosphoproteome in the future (Hogan et al., 2005; McAlister et al., 2007; Hubler et al., 2008; McAlister et al., 2008). Our data clearly illustrate that the study of regulatory phosphorylation in controlling vertebrate development is rich with unexamined questions.

METHODS

Xenopus laevis eggs, oocytes, embryos and gastrula stage modified tissues

Xenopus laevis oocytes, eggs and embryos were generated using standard methods (Lane and Sheets 2000; Mitchell and Sheets 2001; Lane et al., 2004). Two hundred defolliculated oocytes (st. VI), unfertilized eggs, embryos (st. 7 and st. 10+) were flash frozen in liquid nitrogen.

In addition samples of gastrula stage embryos (st. 10+) subjected to several different manipulations were also collected for analysis. These included LiCl treatment to activate the β-catenin pathway and injection of a morpholino to β-catenin to block β-catenin expression and signaling. For LiCl treatment, 32-cell embryos were exposed to 0.3 M LiCl in 0.1x MMR for 12 minutes. After LiCl treatment, embryos were washed four times every 15 minutes with R/10 buffer at pH 7.4. Embryos were allowed to develop to stage 10+ and flash froze in liquid nitrogen. β-catenin injected embryos were prepared by injecting 4-8 cell embryos with 10 ng of β-catenin morpholino (Heasman et al., 2000). These embryos were then allowed to develop to stage 10+, where they were flash frozen in liquid nitrogen. Organizer and non-organizer tissues were prepared by selective tissue enrichment of stage 10+ embryos using a Gastromaster microsurgery instrument. An equivalent of two hundred embryos for each tissue sample were flash frozen for protein extraction.

Protein extraction

Tissue was homogenized in 15 mM Tris pH 8, 75 ug PMSF, 50 mM NaF, 1 mM NaOV, and 25 mM β-glycophosphate. An equal volume of 1,1,2-trichlorotrifluoroethane (Freon) was mixed with homogenate, centrifuged and the lower phase containing yolk proteins removed. The sample was mixed with Trizol (400 μl), incubated for 3 minutes at room temperature and separated by centrifugation. Isopropanol was added to the bottom phase and incubated for 10 minutes to precipitate the proteins, and centrifuged to pellet the proteins. The pellet was washed three times with 0.3 M guanidine-HCl (in 95% ethanol), washed once with 100% ethanol and dried to remove residual ethanol. The pellet was resuspended in 6 M guanidine-HCl, 25 mM NH4HCO3, 50 mM NaF, 1 mM NaOV, and 25 mM β-glycophosphate. A buffer exchange using 6 M urea followed by 25 mM ammonium bicarbonate (pH 8) was done to lower guanidine hydrochloride concentration to 0.1 M, and the urea concentration to 2 M.

Phosphopeptide enrichment

Extracted proteins were prepared for proteolysis by reducing with 500 uM DTT for one hour, 1.2 mM iodo-acetate for 45 minutes in the dark followed with 1.25 mM DTT. The proteins were then treated with sequencing grade trypsin for one hour at 37 °C and quenched with glacial acetic acid (pH 3). The sample was desalted using a C18 column, and the peptide containing eluent brought to dryness using a vacuum centrifuge. Esterification of peptides was completed by adding 40μl of thionyl chloride to 1 ml of anhydrous methanol and adding this solution to the dried peptides for 1 hr with mixing at room temperature. The modified peptides were brought to dryness again, and resuspended in a reconstitution buffer of equal parts methanol, acetonitrile, and water, with a 0.01% acetic acid concentration. Modified peptides were loaded onto a 560 μm I.D. × 8 cm fused-silica column packed with Poros MC particles (Applied Biosystems) activated with 100 mM FeCl3. Non-phosphorylated peptides were removed by rinsing the column with the reconstitution buffer. Captured phosphopeptides were then eluted onto a nHPLC pre-column (360 μm × 75 μm, packed with 10-30 μm C8 particles) using 15 μL of 50 mM phosphate buffer, pH 8.

LC-MS/MS

The pre-column was connected to an nHPLC analytical column (360 μm o.d. × 50 μm i.d.) packed with 5 cm of 5μm C8 reversed-phase packing material and equipped with an integrated, electrospray emitter tip (Martin et al. 2000). Peptides were eluted into a Thermo LTQ mass spectrometer modified to perform electron transfer dissociation (ETD) (Coon et al., 2004; Syka et al., 2004). Peptides were eluted at a flow rate of 60 nL/min with the following gradient: 0-7% B in 5 min, 7% - 45% B in 70 min, 45-100% in 15 min, 100-0% B in 5 min, (A = 0.1 M aqueous acetic acid, B = 70% acetonitrile in 0.1 M aqueous acetic acid). The mass spectrometer was set to continuously cycle through the acquisition of a MS1 spectra from which the 10 most abundant precursors were automatically selected for tandem MS interrogation via ETD (Haas et al., 2006).

LiCl treated and β-catenin morpholino injected embryos were prepared and analyzed in a consistent manner with protocol above, with the exception that the LiCl treated samples were analyzed by ETD only, and the β-catenin morpholino injected samples were analyzed by ETD and collision activated dissociation (CAD) on a hybrid linear ion trap-orbitrap mass spectrometer (McAlister et al., 2007).

Data analysis

All tandem mass spectra were searched against the Xenopus laevis subsection of the NCBI database using the Open Mass Spectrometry Search Algorithm (OMSSA). Static modifications of 58.01 Da on cysteine (carbamidomethylation), and 14.02 Da on glutamic acid, aspartic acid, and the peptide c-terminus to account for the o-methyl ester modification. Dynamic modifications of +79.9663 Da at serine, threonine, and tyrosine residues for phosphorylation, and +16 Da at methionine for oxidation were also employed. Confidence values were assessed by a target-decoy search strategy (Käll et al., 2008) to identify phosphopeptides in two groups (<0.5% false positive rate (FPR), and <5% FPR). Identified peptides were grouped into 12 functional categories according to their predicted parent protein according to accession number using a functional annotation database clustering program called Database for Annotation Visualization and Integrated Discovery (DAVID) available at (http://david.abcc.ncifcrf.gov/home.jsp).

Immunoblotting

Total protein from equivalent numbers of Xenopus oocytes eggs, stage 7 embryos and stage 10+ embryos were loaded onto each lane and electrophoresed on 12.5% SDS-polyacrylamide gels. Proteins were transferred to PVDF membrane, the membrane was blocked and probed with an anti-4E-BP2 antibody that detects 4E-BP2 when it is phosphorylated at Thr-33 or Thr-44 (Cell Signaling, phospho-4E-BP1 (Thr37/46) #2855). The membrane was incubated with goat anti-rabbit antibodies coupled to horseradish peroxidase. Antibody binding was detected using a chemiluminescent substrate and exposure to x-ray film. Antibodies were stripped from the filter by incubation in a solution of 0.2 M Glycine-HCl pH2.5, 0.05% Tween-20, 100 mM 2-mercaptoethanol for 60 min. The same filter was probed with a second anti-4E-BP2 antibody that detects total 4E-BP2 regardless of its phosphorylation state (Santa Cruz). Antibody binding was detected as described above.

EST cluster comparison

Seventy Xenopus laevis gastrula phosphoproteins were randomly chosen for analysis from SI. Tables 2, 3 and 4; APC-binding protein (EB2) (NP_001083449), Ataxin2 (AAH97692), β-catenin (AAI08765), Brg1 (AAX08100), BRM (AAQ02780), Interleukin Enhancer Binding Factor 3 (NP_001083930), Casein Kinase II (Beta) (P28021), Cnot2 (AAH73075), Denticleless (AAH73015), ELAV (NP_001081035), enhancer of zeste-2 (EZH2) (AAK30208), Eyes absent-3 (NP_001089994), FOXD3a (Q9DEN4), FoxN3a (NP_001090178), Fzd7 (AAH42228), ID2 (BAA76634), ISWI (imatation switch) (AAG01537), Jumonji (AAH88951), Lethal giant larvae 2 (AAH72924), Lin-28 (AAH42225), Lunitic fringe (AAI29635), MASKIN (AAF19726.1), Mastermind1 (NP_001080927), Mi2 deacetylase (AAD55392.1), mRNA-4 p56 (FRGY2) (P21574), Poly (A) binding protein-1 (AAO33927), Programmed cell death 4 (AAH56125), Pumilio (AAL14121), RAP55 (AAH42251), Ski2 (NP_001084112), Sox3 (AAH72222), Tra-2 (AAH44990), β-TRCP (AAA02810), Treacle (AAW56574), V-ets (AAI55947), Vg1 RNA binding protein (AAC18597), Xeek1 (AAC59904), zinc finger protein 36 (AAH84221), zinc finger transcription factor SALL4 (AAH95923), Api5 (AAH77529), death associated protein (AAI33257), deoxyuridine triphosphatase 3 (NP_0001091360), echinoderm microtubule associated protein 4 (AAI10955), eukaryotic translation elongation factor 2 (EF-2) (AAH44327), eukaryotic translation initiation factor 4 gamma (EIF4G) (AAH89197), eukaryotic initiation factor 4E binding protein 2 (4E-BP2) AAI33809, heat shock protein (90kDa) NP_001086624, high mobility group AT-hook 2 (AAI24963), histone binding protein N1/N2 (CAA28419), insulin-like growth factor 2 mRNA binding protein 1 (AAH57700), NDRG (NP_001080389), NOL1/NOP2/Sun domain 2 (Nsun2) (AAH68818), Nsfl1 (p47) (AAH41297), Nucleoplasmin (CAA28460), PAICS (AAH41276), pyruvate dehydrogenase alpha (PDHA) (AAI06671), programmed cell death 4 (AAH56125), RAD23 (AAH44115), Ribosomal protein L15 (AAH46569), Ribosomal protein L34 (AAH99259), Ribosomal protein S6 (AAH54151), serpin (AAA49703), Ribosomal protein L28 (AAH53798), stathmin 1/oncoprotein 18 (NP_001080672), eukaryotic translation initiation factor 4H (EiF4H) (NP_001083502), MCM4 (AAH83031), nuclear autoantigenic sperm protein (NASP1) (NP_001089907), nucleolin (AAI57416), nucleoporin (CAB53357), Ribosomal protein L24 (AAH78474), Ribosomal protein S1a (CAA40592), Ribosomal protein L13 (AAH75140), Upstream of NRAS (NP_001090026), CIRP2 (AAH54250), eukaryotic translation initiation factor 4E nuclear import factor 1 (4E-T) (NP_001086710). For each Xenopus laevis phosphoprotein, the analogous Xenopus tropicalis mRNA was identified along with the associated ESTs. The Xenopus tropicalis ESTs for specific mRNAs have been sorted into clusters where the number of ESTs per cluster provides an estimate of mRNA expression levels (http://www.sanger.ac.uk/Projects/X_tropicalis/) (Gilchrist et al., 2004) EST clusters were identified for each mRNA corresponding to a Xenopus laevis phosphoprotein. The number of ESTs per cluster was compared to the number of phosphoprotein encoded mRNAs that map to each cluster and plotted.

Supplementary Material

SI Table 1

SI Table 2

SI Table 3

4

Supplementary Information (SI):

SI. Table 1. Xenopus phosphorylation events. The total results from the Xenopus phosphoproteomic analysis as summarized in Table 1. In addition, this table also contains results from the analysis of gastrula stage embryos manipulated in different ways, such as LiCl treatment or embryos injected at the 2 cell stage with β-catenin morpholino.

SI. Table 2. Phosphorylation events found at all stage of development examined. The results from the Xenopus phosphoproteomic analysis (Sup. Table 1) were examined to identify phosphorylated sites present in at least three of the four stages of development analyzed. The first column contains the peptide fragment identified with the phosphorylation site in lowercase. Also shown are the start and end points of the peptide relative to the full-length protein it was derived from (the accession number is also listed along with the protein name or Unigene identifier if the protein was unidentified in Xenopus laevis).

SI. Table 3. Phosphorylation of proteins relevant to embryonic development. The results from the Xenopus phosphoproteomic analysis (Sup. Table 1) were examined to identify phosphorylated sites present on proteins of interest during the four stages of development analyzed. The first column contains the peptide fragment identified with the phosphorylation site in lowercase. Also shown are the start and end points of the peptide relative to the full-length protein it was derived from (the accession number is also listed along with the protein name or Unigene identifier if the protein was unidentified in Xenopus laevis).

ACKNOWLEDGMENTS

We thank CA Fox and Y Zhang for comments on the manuscript. We also acknowledge the University of Wisconsin-Madison, Thermo Scientific, the Beckman Foundation, the American Society of Mass Spectrometry, Eli Lilly, the National Science Foundation (0701846; 0747990 to JJC), and the NIH (Grant R01-HD43996 to MDS and R01GM080148 to JJC) for support of this work. DLS gratefully acknowledges support from an NIH pre-doctoral fellowship – the Genomic Sciences Training Program, NIH 5T32HG002706 for support of this work.

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